Genetic engineering has allowed the improvement of microorganisms used as industrial bioreactors, cell factories and in food fermentations. The Bacillus genera produce and secrete a large number of useful proteins and metabolites (Zukowski 1992 Zukowski M M (1992) Production of commercially valuable products. In: Doi R H, McGlouglin M (eds) Biology of bacilli: applications to industry. Butterworth-Heinemann, Stoneham. Mass. pp 311-337). The most common bacilli used in industry are B. licheniformis, B. amyloliquefaciens and B. subtilis. Furthermore, because of its GRAS (generally recognized as safe) status, B. subtilis is a natural candidate for the production of proteins utilized in the food and pharmaceutical industries.
The aprE gene of B. subtilis codes for the extracellular protease subtilisn, a valuable enzyme manufactured by the biotechnology industry (Debadov V G (1982) The Industrial Use of Bacilli. In: Dubnau D A (ed) The Molecular Biology of the Bacilli. Academic Press: New York/London, vol 1, pp 331-370). The development of recombinant protein production systems using B. subtilis as a host organism, especially those driven by the subtilisin promoter, provides an important tool for research and commercial production in this area (Oyama et al. (1989) Secretion of Escherichia coli Aminopeptidase P in Bacillus subtilis using the Prepro-Structure Coding Region of Subtilisin Amylosacchariticus. J. Ferment. Bioeng. 68: 289-292). Although subtilisin synthesis is not required for sporulation (Stahl and Ferrari (1984) Replacement of the Bacillus subtilis Subtilisin Structural Gene With an In Vitro-Derived Deletion Mutation. J Bacteriol. 158: 411-418), its production is triggered by mechanisms common to those events responsible for the sporulation initiation, and hence, it has served as a model for developmentally-associated gene expression (Sonenshein A L (1989) Metabolic Regulation of Sporulation and Other Stationary-Phase Phenomenon. In: Smith I, Slepecky R A, Setlow P (eds) Regulation of Procaryotic Development. American Society for Microbiology, Washington, D.C. pp 109-130). The aprE gene is transcribed by sigma A (σA) and its expression is highly controlled by several regulators, such as: DegU/DegS, AbrB, Hpr and SinR (Valle and Ferrari (1989) In: Smith I, Slepecky R A, Setlow P (eds) Regulation of Procaryotic Development. American Society for Microbiology. Washington, D.C. pp 131-146). A consensus sigma A promoter has been identified (Helman et al., 1995, Nucleic Acid Research, Vol. 23, pp. 2351-2360). In spite of advances in the understanding of production of proteins in host cells, there remains a need for methods for increasing expression of proteins in host cells, such as Bacillus host cells.